The human immunodeficiency virus, HIV, encodes three key viral enzymes through its pol gene and these enzymes are critical for the replication of this virus [Fauci, Science, 239, 617-622 (1988); Katz & Skalka, Annu. Rev. Biochem., 63, 133-173 (1994); Frankel, Annu. Rev. Biochem., 67, 1-25 (1998)]. For this reason, these enzymes of the pol gene have been targeted as potential sites of attack in the development of HIV antiviral chemotherapeutic agents [De Clercq, J. Med. Chem. 38, 2491-2517 (1995); Clin. Microbiol. Rev., 10, 674-693 (1997); De Clercq, Nature Reviews: Drug Discovery, 11, 13-25 (2002)]. Drug discovery involving two of these enzymes, HIV reverse transcriptase (RT) and HIV protease (PR), and subsequent clinical applications of some of these therapeutic agents in combination therapy for the treatment of acquired immunodeficiency syndrome (AIDS) and AIDS related complex (ARC) in HAART (highly-active antiretroviral therapy) have suggested that this methodology of targeting key viral enzymes represents a useful approach in antiviral chemotherapy [Johnson & Gerber, in “Advances in Internal Medicine,” vol. 44. Mosby: St. Louis, 1-40 (2000); De Clercq, Nature Reviews: Drug Discovery, 11, 13-25 (2002); Miller & Hazuda, Current Opinion in Microbiology, 4, 535-539 (2001); Asante-Appiah & Skalka, Adv. virus Res., 52, 351-369 (1999); Nair, in “Recent Advances in Nucleosides: Chemistry and Chemotherapy,” Elsevier Science: Netherlands, 149-166 (2002); DeClercq, Intl. J. Biochem. Cell Biol. 36, 1800-1822 (2004)]. While HIV RT and HIV PR have been extensively studied with respect to therapeutics, the third enzyme of the pol gene, HIV integrase, has received much less consideration [Miller & Hazuda, Current Opinion in Microbiology, 4, 535-539 (2001); Nair, Rev. Med Virol., 12, 179-193 (2002); Nair, Current Pharmaceutical Design, 9, 2553-2565 (2003)].
At present there are no drugs in clinical use for HIV/AIDS where the mechanism of action is inhibition of HIV integrase. HIV-1 integrase is a protein of 32 kDa encoded at the 3′-end of the pol gene [Asante-Appiah & Skalka. Adv. Virus Res., 52, 351-369 (1999); Esposito & Craigie, Adv. Virus Res., 52, 319-333 (1999)]. It is involved in the integration of HIV DNA into the host cell chromosome. Because integrase has no human counterpart and because it plays the significant role of completing the invasion of the human cell by HIV, it is an attractive target for the discover), of inhibitors of therapeutic potential.
Incorporation of HIV DNA into host chromosomal DNA in the cell nucleus catalyzed by integrase apparently occurs by a specifically defined sequence of 3′-processing or tailoring and strand transfer/integration reactions [Asante-Appiah & Skalka, Adv. Virus Res., 52, 351-369 (1999); Esposito & Craigie Adv. Virus Res., 52, 319-333 (1999)]. Prior to the initiation of the integration process, there is assembly of viral DNA, previously produced by reverse transcription, on the integrase. HIV integrase recognizes specific sequences in the LTRs of viral DNA. Following assembly of viral DNA on integrase, the processing of viral DNA occurs where there is site specific endonuclease activity and two nucleotides are cleaved off from each 3′-end of the double helical viral DNA to produce the tailored viral DNA recessed by two nucleotides and bearing a terminal CAOH-3′. For this initial 3′-processing step, integrase apparently activates the phosphodiester bond towards cleavage. The recessed viral DNA thus produced is joined in the next step to host cell DNA in the nucleus through a trans-esterification reaction. In this step, integrase positions the 3′-OH end of the viral DNA for nucleophilic attack on the phosphodiester bond in the host DNA. In the subsequent step, there is cleavage of 4-6 bp in host DNA and the coupling involves the joining of processed CAOOH-3′ viral DNA ends to the 5′-phosphate ends of the host DNA. Finally, there is repair of the resulting gapped intermediate mediated by host cell enzymes, although a role here for the integrase is also possible.
A variety of compounds are inhibitors of HIV integrase but some of these compounds are non-specific inhibitors of the enzyme while evidence suggests that others may possess some specificity. The various classes include nucleotides, oligonucleotides, dinucleotides, and miscellaneous small molecules including heterocyclic systems, natural products, diketo acids, sulfones and others [Nair, Rev. Med. Virol., 12, 179-193 (2002); Nair, Current Pharmaceutical Design, 9, 2553-2565 (2003); Chi and Nair, Bioorg. Med. Chem. Lett. 14, 4815-4817 (2004); Nair and coworkers, J. Am. Chem. Soc., 122, 5671-5677 (2000)].
The class of previously studied compounds that are most directly relevant to this patent are diketo acids with aryl or heteroaryl substitutions. Some of these compounds are inhibitors of HIV integrase, but most commonly of only the strand transfer step. The integrase inhibition data have been reported in several scientific publications [Wai, et al., “4-Aryl-2,4-dioxobutanoic acid inhibitors of HIV-1 integrase and viral replication in cells,” J. Med. Chem. 43, 4923-4926 (2000); Pais, G. C. C., et al., “Structure activity of 3-aryl-1,3-diketo-containing compounds as HIV-1 integrase inhibitors.” J. Med. Chem. 45, 3184-3194 (2002); Marchand, C., et al., “Structural determinants for HIV-1 integrase inhibition by β-diketo acids,” J. Biol. Chem. 277, 12596-12603 (2002); Sechi, M., et al., “Design and synthesis of novel indole beta-diketo acid derivatives as HIV-1 integrase inhibitors,” J. Med. Chem. 47, 5298-5310 (2004); Zhang, et al., “Azido-containing aryl β-keto acid HIV-1 integrase inhibitors,” Bioorg. Med. Chem. Lett. 13, 1215-1219 (2003)]. Other publications in the area are of peripheral relationship to this patent application.
The mechanism of inhibition of HIV integrase by diketo acids may be the result of interaction of the functional groups on these compounds with metal ions in the active site of integrase, resulting in a functional sequestration of these critical metal cofactors [Grobler, J. A., et al., Proc. Natl. Acad. Sci. U.S.A. 99, 6661-6666 (2002)].
The most directly related patents to this application are: Selnick, H. G. et al., (Merck & Co. Ltd.), “Preparation of nitrogen-containing 4-heteroaryl-2,4-dioxobutyric acids useful as HIV integrase inhibitors,” WO 9962513; Young, S. D., et al., (Merck & Co. Ltd.), “Preparation of aromatic and heteroaromatic 4-aryl-2,4-dioxobutyric acid derivatives useful as HIV integrase inhibitors,” WO 9962897; Fujishita, T., et al., Yoshinaga, T., et al. (Shionogi & Co. Ltd.), “Preparation of aromatic heterocycle compounds having HIV integrase inhibiting activities,” WO 0039086; Akihiko, S., (Shionogi & Co. Ltd.), “Medicinal compositions containing propenone derivatives,” WO 0196329; Payne, L. S., et al., (Merck & Co. Ltd.; Tularik, Inc.), “Preparation of 1,3-diaryl-1,3-propanediones as HIV integrase inhibitors,” WO 0100578; Egbertson, M., et al., (Merck & Co. Ltd.), “HIV integrase inhibitors,” WO 9962520.
Some of the patents cited above are closely related. However, none of the patents or publications describe the class of compounds according to the present invention. In particular, compounds according to the present invention have a nucleic acid base (nucleobase) scaffold, which is a requirement for potent activity and which was not previously recognized.
Structures of the three most active compounds described in previous patents and publications are shown below. They are representative of the best of the previously known inhibitors. They inhibit the strand transfer step in the HIV integrase assay but do not show significant inhibition of the 3′-processing step of integrase action.
In vitro anti-HIV activity data Compd. No:S-1360L-731988L-708906IC50 (μM):   0.14*1.0 5.5**CC50 (μM):110*Not Given88.3**Therapeutic Index786*Not Given16** Patents:WO 0196329WO 9962513WO 9962520Publications:Hazuda, D. J., et al.,Hazuda, D. J., et al.,Science 287, 646-650Science 287, 646-650(2000)(2000)*Anti-HIV-1 data of Yoshinaga et al from Proc. 9th Conference on Retroviruses and Opportunistic Infections, 2002 (no 8, p 55) cited by DeClercq, Int. J. Biochem. &  Cell Biol., 36, 1800-1822 (2004).**These in vitro anti-HIV-1 data were reported for compound L-708906 by the DeClercq laboratory [Pannecouque, C., et al., Current Biology, 12, 1169-1177 (2002)].
However, the class of compounds described by us in this invention is not only significant because of the strong inhibition of both the 3′-processing and strand transfer steps of HIV integrase, but also because of its enhanced potency and therapeutic index as evidenced by in vitro anti-HIV activity. Critical in the design of our compounds is the nucleic acid base (nucleobase) scaffold, which is a requirement for potent activity and which was not previously recognized.
As an example, in a side by side in vitro anti-HIV activity comparison with AZT (PBMC cell-based assay), the compound of the present invention shown below with a uracil scaffold (see also general formula I) had a therapeutic index of >10,000 and was more active than AZT (therapeutic index=5,511). This compound is also far more active than the anti-HIV integrase inhibitors previously described in the publications and patents cited above. The therapeutic index of the best known HIV integrase inhibitor previously reported (see compound number S-1360 in Table above) in vitro anti-HIV assays was only 786.